2.1. Antimicrobial Agents
Infectious diseases sicken or kill millions of people each year. Each year in the United States alone, hundreds of thousands of people are infected with resistant bacterial strains that are no longer treatable with drugs like penicillin and vancomycin (Hiramatsu et al, 1997, Morbidity and Mortality Weekly Report 46:624-26). Infections associated with antimicrobial resistance include those acquired in hospitals (nosocomial), such as pneumonia particularly in the young, elderly and immunocompromised), typhoid fever, bacterial meningitis, and tuberculosis. Around the world, nearly 1.5 billion people carry various types of the tuberculosis bacteria and depending on the country, up to 40 percent have proven to be resistant to antibiotics (see, Boyce et al, 1997, Epidemilogy and prevention of nosocomil infections. In The Staphylococcus in Human Disease. Crossley and Archer Eds, Chruchill Livingston Inc., New York, N.Y.). It is estimated that in some developed countries, up to 60% of all nosocomial infections result from bacteria resistant to antibiotics. For example, Pseudomonas aeruginosa, is of the most common gram-negative bacterium associated with nosocomial infections and outbreaks in burn units. Infections by this organism are associated with high mortality (60%), which is attributed to the high intrinsic resistance of members of this genus to many structurally unrelated antibiotics. Gram-positive bacteria also have a significant impact on infectious diseases. For example, Staphylococcus aureus, is a Gram-positive organism which is responsible for about 260,000 hospital acquired infections in the United States which subsequently causes between 60,000 and 80,000 deaths annually (see, Boyce et al, supra).
Although, numerous antimicrobial therapies have been designed to target one or several infectious agents, many therapies show varying degrees of success in eradicating infection. Only a very limited number of new antibiotics have come onto the market in the last decade, yet the number of deadly bacteria that are resistant to these drug therapies has soared. For example, vancomycin is one of the last effective antimicrobial available for the treatment of methicillin-resistant S. aureus infection (MRSA). However, vancomycin resistant isolates S. aureus have now emerged (Hiramatsu et al, 1997, Morbidity and Mortality Weekly Report 46:624-26). Additionally, the failure of many of these therapies to target specific infectious agents has lead to overuse or inappropriate use of the therapies, which in turn has lead to the development of drug resistant microbes. The development of drug resistance in many infectious agents has reduced the efficacy and increased the risk of using the traditional antimicrobial therapies.
Accordingly, there is need in the art for novel molecules and novel combinations of molecules that can act as lethal agents in bacteria and which may be delivered to a pathogen, without causing toxicity to the infected host. Further, there is a need in the art for novel methods of targeting particular species of pathogens while leaving the host's beneficial flora intact. The present invention provides such novel products, therapeutics, and methods for delivery which may be used as toxic agents against pathogens such as bacteria.
2.2. Antisense
Antisense technology seeks to use RNA molecules which are complementary to (or antisense to) a cellular RNA, for the purpose of inhibiting a cellular RNA from being translated into the encoded protein. In this way, the expression of a specific protein is targeted for down regulation. However, a large number of difficulties exist in the art surrounding antisense technology. Commonly, delivery of an exogenous antisense molecule to the target cell is difficult or impossible to achieve. Further, antisense molecules do not consistently lead to a decrease in protein expression. For example, it has been shown that the expression of antisense RNA in transgenic mice did not invariably lead to a reduction in target RNA molecules, and when reduction in target RNA molecules did occur, it was not predictably paralleled by a reduction in protein. Even when protein levels were reduced sometimes no biological effect was detected (Whitton, J. Lindsay “Antisense Treatment of Viral Infection” Adv. in Virus Res. Vol. 44, 1994). Thus, there is a need in the art for a delivery system in which antisense molecules may be efficiently delivered to a target cell such as a bacterial pathogen.
2.3. Ribozymes
A ribozyme is a catalytic RNA molecule that cleaves RNA in a sequence specific manner. A key technical concern in the use of ribozymes as antimicrobial agents is that the ribozyme must be introduced into and expressed by the targeted microbe so that the ribozyme(s) can cleave the targeted RNA(s) inside the microorganism. A second important concern is the tight coupling of transcription and translation in microorganisms which can prevent binding to and cleavage of the bacterial RNA targets. Additionally, bacterial RNAs often have a shorter half life than eukaryotic RNAs, thus lessening the time in which to target a bacterial RNA. The invention described herein addresses these concerns and proves novel therapeutic treatments of bacterial infections using combinations of ribozymes and toxic agents.